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      RNA-Seq Approach for Genetic Improvement of Meat Quality in Pig and Evolutionary Insight into the Substrate Specificity of Animal Carbonyl Reductases

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          Abstract

          Changes in meat quality traits are strongly associated with alterations in postmortem metabolism which depend on genetic variations, especially nonsynonymous single nucleotide variations (nsSNVs) having critical effects on protein structure and function. To selectively identify metabolism-related nsSNVs, next-generation transcriptome sequencing (RNA-Seq) was carried out using RNAs from porcine liver, which contains a diverse range of metabolic enzymes. The multiplex SNV genotyping analysis showed that various metabolism-related genes had different nsSNV alleles. Moreover, many nsSNVs were significantly associated with multiple meat quality traits. Particularly, ch7:g.22112616A>G SNV was identified to create a single amino acid change (Thr/Ala) at the 145th residue of H1.3-like protein, very close to the putative 147th threonine phosphorylation site, suggesting that the nsSNV may affect multiple meat quality traits by affecting the epigenetic regulation of postmortem metabolism-related gene expression. Besides, one nonsynonymous variation, probably generated by gene duplication, led to a stop signal in porcine testicular carbonyl reductase (PTCR), resulting in a C-terminal (E281-A288) deletion. Molecular docking and energy minimization calculations indicated that the binding affinity of wild-type PTCR to 5α-DHT, a C 21-steroid, was superior to that of C-terminal-deleted PTCR or human carbonyl reductase, which was very consistent with experimental data, reported previously. Furthermore, P284 was identified as an important residue mediating the specific interaction between PTCR and 5α-DHT, and phylogenetic analysis showed that P284 is an evolutionarily conserved residue among animal carbonyl reductases, which suggests that the C-terminal tails of these reductases may have evolved under evolutionary pressure to increase the substrate specificity for C 21-steroids and facilitate metabolic adaptation. Altogether, our RNA-Seq revealed that selective nsSNVs were associated with meat quality traits that could be useful for successful marker-assisted selection in pigs and also represents a useful resource to enhance understanding of protein folding, substrate specificity, and the evolution of enzymes such as carbonyl reductase.

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          Differences in gene expression may play a major role in speciation and phenotypic diversity. We examined genome-wide differences in transcription factor (TF) binding in several humans and a single chimpanzee by using chromatin immunoprecipitation followed by sequencing. The binding sites of RNA polymerase II (PolII) and a key regulator of immune responses, nuclear factor kappaB (p65), were mapped in 10 lymphoblastoid cell lines, and 25 and 7.5% of the respective binding regions were found to differ between individuals. Binding differences were frequently associated with single-nucleotide polymorphisms and genomic structural variants, and these differences were often correlated with differences in gene expression, suggesting functional consequences of binding variation. Furthermore, comparing PolII binding between humans and chimpanzee suggests extensive divergence in TF binding. Our results indicate that many differences in individuals and species occur at the level of TF binding, and they provide insight into the genetic events responsible for these differences.
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            Resequencing of 200 human exomes identifies an excess of low-frequency non-synonymous coding variants.

            Targeted capture combined with massively parallel exome sequencing is a promising approach to identify genetic variants implicated in human traits. We report exome sequencing of 200 individuals from Denmark with targeted capture of 18,654 coding genes and sequence coverage of each individual exome at an average depth of 12-fold. On average, about 95% of the target regions were covered by at least one read. We identified 121,870 SNPs in the sample population, including 53,081 coding SNPs (cSNPs). Using a statistical method for SNP calling and an estimation of allelic frequencies based on our population data, we derived the allele frequency spectrum of cSNPs with a minor allele frequency greater than 0.02. We identified a 1.8-fold excess of deleterious, non-syonomyous cSNPs over synonymous cSNPs in the low-frequency range (minor allele frequencies between 2% and 5%). This excess was more pronounced for X-linked SNPs, suggesting that deleterious substitutions are primarily recessive.
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              We have developed a formalism and a computational method for analyzing the potential functional consequences of non-synonymous single nucleotide polymorphisms. Our approach uses a structural model and phylogenetic information to derive a selection of structure and sequence-based features serving as indicators of an amino acid polymorphim's effect on function. The feature values can be integrated into a probabilistic assessment of whether an amino acid polymorphism will affect the function or stability of a target protein. The method has been validated with data sets of unbiased mutations in the lac repressor and lysoyzyme. Applying our methodology to recent surveys of genetic variation in the coding regions of clinically important genes, we estimate that approximately 26-32 % of the natural non-synonymous single nucleotide polymorphisms have effects on function. This estimate suggests that a typical person will have about 6240-12,800 heterozygous loci that encode proteins with functional variation due to natural amino acid polymorphism. Copyright 2001 Academic Press.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                4 September 2012
                : 7
                : 9
                : e42198
                Affiliations
                [1 ]Department of Animal Resources Technology, Gyeongnam National University of Science and Technology, Jinju, Korea
                [2 ]Swine Science and Technology Center, Gyeongnam National University of Science and Technology, Jinju, Korea
                [3 ]SeqGenesis, Daejeon, Korea
                [4 ]Division of Applied Life Science (BK21 Program), Gyeongsang National University, Jinju, Korea
                [5 ]Plant Molecular Biology and Biotechnology Research Center (PMBBRC), Gyeongsang National University, Jinju, Korea
                [6 ]Da-San-Jong-Don Co. Ltd., Namwon, Korea
                [7 ]National Institute of Animal Science, RDA, Suwon, Korea
                [8 ]Division of Life Science, Korea Basic Science Institute, Daejeon, Korea
                [9 ]Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, Korea
                University of Queensland, Australia
                Author notes

                Competing Interests: The authors have read the journal's policy and have the following conflict: Hwa Choon Park and both Won Yong Jung and Seung-Won Lee are employed by Da-San-Jong-Don Co. Ltd. and SeqGenesis, respectively. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials. Except for these three authors, the rest of the authors in this manuscript have declared that no conflict of interest exists.

                Conceived and designed the experiments: KWL WYB CWK. Performed the experiments: ESC SGK MS YL BWK DHP KKG TWK DHK SKJ ISK YMS KHC JHH JHK. Analyzed the data: BYP JSC WYB SWL WYJ KKC. Contributed reagents/materials/analysis tools: HCP. Wrote the paper: WYB CWK.

                Article
                PONE-D-11-14356
                10.1371/journal.pone.0042198
                3433470
                22962580
                5079a0c2-350c-467f-84fd-4f759b7dc96f
                Copyright @ 2012

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 26 July 2011
                : 5 July 2012
                Page count
                Pages: 11
                Funding
                This work was supported by grants from the Priority Research Centers Program (2011-0022965) and Basic Science Research Program (2012-0008319) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology, and the Technology Development Program for Agriculture and Forestry (107091-5), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea. It was also carried out with the support of “Cooperative Research Program for Agriculture Science and Technology Development (PJ0077672011)” Rural Development Administration, Republic of Korea. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Agriculture
                Animal Management
                Animal Breeding
                Animal Genetics
                Animal Production
                Biology
                Computational Biology
                Macromolecular Structure Analysis
                Protein Structure
                Molecular Genetics
                Population Genetics
                Genetics
                Population Genetics
                Genetic Polymorphism
                Medicine
                Diagnostic Medicine
                Pathology
                General Pathology
                Biomarkers

                Uncategorized
                Uncategorized

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